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2.7.1 Preparation of DNA probes for hybridisation

2.7.1.1 DNA labelling

For molecular hybridisation, DNA template was randomly labelled with radioactive α-

32

P dCTP by the kit of Prime-a-Gene labelling system of Promega, Mannheim, according to the user manual briefly described as below.

Thaw all the system components except the Klenow fragment on ice. Add xµl (~25ng) DNA template into a 1.5ml Eppendorf tube containing (33-x)µl DNase-free H2O, and

denature at 95~100°C for 2 min, then chill the tube rapidly on ice for 5 min. Afterwards, add the other components of the following reaction setting in the tube (on ice) with the DNA template.

Denatured DNA template(~25ng)...x µl (in the tube) 5x labelling buffer...10 µl Mixture of dNTPs: 1.5mM dATP...0.667 µl 1.5mM dGTP...0.667 µl 1.5mM dTTP...0.667 µl Nuclease-free BSA(10mg/ml)...2 µl α-32P dCTP, 50µCi, 3000Ci/mmol (red)...2 µl DNA polymerase I, klenow large fragment(5U/µl)...1 µl DNase-free H2O...33-x µl

--- to total volume of 50 µl

Mix the components gently and incubate the reaction tube at room temperature for 1~2 hours.

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2.7.1.2 Purification of the labelled DNA probe

After the labelling reaction, the labelled DNA was purified by Microspin columns of Amersham, Braunschweig, to remove short-sized labelled DNA fragments and unincorporated nucleotides by the following procedure. Resuspend the resin in the column by vortexing vigorously for 1min. Loosen the cap one-fourth turn and snap off the bottom closure, place in a 1.5ml Eppendorf tube and spin the column for 1 min at 3000 rpm. Put the column in a new 1.5ml Eppendorf collection tube, open the cap of the column and slowly apply the labelling mixture to the top centre of the resin without disturbing its bed, cover the tube with the cap loosely, then spin the column at 3000rpm for 2 min. The flow-through in the collection tube is about 50µl and colourless, while the red unincorporated nucleotides and short-labelled fragments remain in the resin of the column. Before hybridisation, denature the probe in a 95~100°C water-bath for 2~3 min, then chill it on ice quickly.

2.7.2 Southern blotting

2.7.2.1 Solutions

- Hybridisation buffer (Church buffer) (Church and Gilbert, 1984):

Na2HPO4/NaH2PO4(PH7.5)...250mM

SDS...7%(w/v)

- Transfer buffer: 0.4N NaOH

- Washing buffer: 0.25x SSC, 0.1% SDS - 20x SSC

- 10% SDS

2.7.2.2 Enzyme digestion of plant DNA

Harvest the leaves from transplastomic plants grown in the culture chamber. Extract the total DNA by Qiagen DNeasy plant Mini kit. Take about 2µg total DNA of each sample for complete digestion with enzyme BglII at 37°C, 3~4 hours or overnight.

2.7.2.3 DNA gel electrophoresis and blotting

Prepare a large 1% agarose gel of the size 20cm x 20cm containing 0.5µg/ml ethidium bromide for southern blotting. Load the DNA digestion sample in a mixture with 6x DNA loading buffer in the gel slot and run at 30~40V overnight for optimal separation, then document the gel with a transparent ruler under UV illumination. Wash the gel with distilled

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water after an optional treatment by 0.25M HCl and assemble the DNA blot unit containing the transfer buffer (0.4N NaOH) in the meantime. Apply the gel on the DNA blot unit to transfer DNA from the gel to the Hybond-N+ nylon membrane (Amersham, Braunschweig) by capillary action. After12~18 hours or overnight, separate the DNA filter, wash in 2xSSC optionally and air-dry for half hour on 3M filter paper, then expose in the UV crossinglinker (Strategene, Heidelberg) for DNA fixation. After that, wrap the DNA filter in 3M filter paper and keep at room temperature till its use for hybridisation.

2.7.2.4 Prehybridisation and hybridisation

Roll the DNA filter and transfer to a glass tube with a thermostable plastic cap. Add proper amount of hybridisation buffer (~25ml Church buffer for a filter of 20cm x 20cm) to the tube and soak the filter gently, avoiding bubbles. Screw the cap of the tube securely and cling to the low-speed rotor of an oven at 60°C for prehybridisation. Typically perform prehybridisation for 2~4 hours, meanwhile prepare and purify the radioactive probe. Then, renew the old buffer with the same amount of Church buffer pre-warmed at 60°C and pipette the denatured probe into the buffer directly, avoiding contact with the filter. Shake the tube gently and invert for several times to evenly distribute the probe in the buffer, then return to the oven for hybridisation at 60°C overnight.

2.7.2.5 Washing and autoradiography

Discard the hybridisation solution into a 50ml plastic tube for safe disposal. Rinse the DNA filter in 50ml washing buffer, then discard. Add half tube volume of washing buffer in the tube to wash the filter at 60°C in the hybridisation oven for 0.5~1 hour, or immerse the filter in a plastic box containing 200ml washing buffer. Monitor the signal by the radioactivity meter till the optimal washing state, then wrap the filter and place it on the phosphorimager plate for a proper exposure time (1~5h). Automatically develop the hybridisation profile by the phosphorimager (Fujifilm BAS1500).

2.7.2.6 Stripping of DNA probes

In case of using the same DNA filter for more than one hybridisation, carefully keep the filter wet always after the first hybridisation. Remove the old DNA probe from the filter by the DNA stripping solution (0.2x SSC, 0.1% SDS, 0.2M Tris⋅HCl, pH7.5).

Incubate the DNA filter in 100~200ml 0.4N NaOH at 42°C for 5 min with gentle agitation. Rinse the filter briefly with DNA stripping solution, then wash it with the same solution afterwards. Repeat this procedure till no detectable radioactivity is left on the filter.

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Place the DNA filter in the hybridisation tube and proceed with the next hybridisation experiment.

2.7.3 Northern blotting

In RNA analysis such as northern blotting, all hardware and solutions are treated to make them RNase-free.

2.7.3.1 Solutions

- RNase-free H2O

treat the distilled water with 0.05% (v/v) DEPC, mix well and place in the hood overnight and autoclave.

- Hybridisation buffer (Church buffer):

Na2HPO4/NaH2PO4(PH7.5)...250mM

SDS...7%(w/v) treat with 0.1% (v/v) DEPC and autoclave.

- 10x MOPS Buffer

MOPS...200mM NaAc...50mM EDTA...10mM

adjust pH to 7.0 with NaOH, treat with 0.1% (v/v) DEPC and autoclave.

- RNA gel running buffer (1L)

10x MOPS buffer...100ml 37% formaldehyde (12.3M)...20ml RNase-free H2O...880ml - 5x RNA sample loading buffer (1ml)

saturated bromophenol blue solution...1.6µl 500mM EDTA, pH 8.0...8µl 37% formaldehyde (12.3M)...72µl 100% glycerol...200µl ion-free formamide...308.4µl 10x MOPS buffer...400µl add RNase-free H2O to 1ml

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To make a saturated bromophenol blue solution, add solid bromophenol blue into distilled water and mix thoroughly until no more will dissolve. Centrifuge and carefully remove the supernatant to a new collection tube.

- 20x SSC, treat with 0.1% (v/v) DEPC and autoclave.

- 10% SDS

- Washing buffer: 0.1x SSC, 0.1% SDS

2.7.3.2 Preparation of 1.2% formaldehyde RNA gels

agarose...1.2g 10x MOPS...10 ml add RNase-free H2O to 100 ml

Heat in the microwave oven to melt the agarose, cool to 65°C, then add 1.8ml 37% formaldehyde (12.3M) and 1µl ethidium bromide (10mg/ml). Mix thoroughly and pour on the gel support. Equilibrate the gel in gel running buffer for 30 min optionally prior to use.

2.7.3.3 RNA gel electrophoresis and blotting

Load ~2 µg RNA for each sample in the slot of RNA gel, run the gel at 60V for 2~4 hours, then document under UV illumination. Wash the gel with RNase-free H2O for 15 min

twice to remove as much formaldehyde as possible. Meanwhile, assemble the RNA blot unit and fill in the transfer buffer (10x SSC). Apply the gel on the RNA blot unit and transfer RNA from gel to Hybond-N+ nylon membrane (Amersham, Braunschweig) overnight by capillary action. Then, treat the RNA filter in a UV-crossinglinker (Strategene, Heidelberg) for RNA fixation, wrap in 3M filter paper and keep at room-temperature until the hybridisation.

2.7.3.4 Prehybridisation and hybridisation

Roll the RNA filter and transfer to a glass tube with a thermostable plastic cap. Add proper amount of hybridisation buffer (~25ml Church buffer a filter of 20cm x20cm) in the tube carefully and soak the filter gently, avoiding bubbles. Screw the cap of the tube securely and cling to the low-speed rotor of an oven at 65°C for prehybridisation. Typically perform prehybridisation for 2~4 hours, prepare and purify the radioactive probe in the meantime. Then, decant the old buffer and fill in the same amount of Church buffer pre-warmed at 65°C, pipette the denatured probe into the buffer directly, avoiding contact with the filter. Shake the tube gently and invert for several times to evenly distribute the probe in the buffer, then return to the oven for hybridisation at 65°C overnight.

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2.7.3.5 Washing and autoradiography

Discard the hybridisation solution into a 50ml tube for safe disposal. Rinse the RNA filter in 50ml washing buffer, then discard. Add half tube volume of washing buffer in the tube to wash the filter at 65°C in the hybridisation oven for 1~2 hours, or immerse the filter in a plastic box containing 200ml washing buffer. Monitor the signal by the radioactivity meter to determine the optimal washing state, then wrap the filter and place it on the phosphorimager plate for a proper time (1~3h). Automatically develop the hybridisation profile by the phosphorimager (Fujifilm BAS1500).

2.7.3.6 Stripping of DNA probes

In case of hybridising the same RNA filter for more than once with different DNA probes, always be careful not to let the filter dry after the first hybridisation. Strip the old DNA probe from the RNA filter with 0.5% SDS solution.

Pre-heat 150~250ml 0.5% SDS solution to boiling (~100°C). Remove the plastic wrap from the filter and immediately immerse the filter in the boiling solution, continue to boil for 5~10 min. Remove the solution from heat and allow to cool for 10 min. Remove the filter from the solution and check the efficiency of stripping with a radioactivity counter. Repeat the stripping procedure if radioactivity can still be detected, then submit it for the next hybridisation.